The Type VI Secretion System (T6SS) is a recently described secretion system present in nearly one quarter of Gram-negative bacteria including many important human pathogens. Because the T6SS is critical to the virulence of many pathogens of major clinical significance, including Vibrio cholerae, Burkholderia mallei, Salmonella typhimurium, Edwardsiella tarda, Aeromonas hydrophila, enteroaggregative E. coli, and Pseudomonas aeruginosa (which is often resistant to current antibiotics), it represents an attractive and novel target for antibiotic development. To date, no drugs have been developed that target the T6SS. In the case of Francisella tularensis, the highly virulent agent of tularemia and a Tier 1 Select Agent, and the closely related bacterium Francisella novicida, the Francisella Pathogenicity Island (FPI) - encoded T6SS is essential for bacterial virulence and required for altered intracellular trafficking, phagosome escape, and intracellular multiplication. We recently made several important advances that will greatly facilitate identification of inhibitors that block the T6SS of F. novicida, which we employ as a model organism. These advances include (1) identification of environmental triggers for assembly and secretion; (2) development of a split-GFP assay for T6SS assembly; (3) development of a high-throughput assay for secretion; and (4) determination of the structure of the contracted sheath at 3.7 by cryoEM and construction of an atomic model of the apparatus. Fn is particularly attractive as a model organism for these studies because its T6SS has high homology with the T6SS of F. tularensis, with which it shares a similar intracellular lifestyle, and because it has a very streamlined T6SS that is amenable to study with less biological ambiguity and complexity than the T6SS of other pathogens. In this collaborative study involving researchers in a laboratory with years of experience studying intracellular pathogens, including F. tularensis, and the Director of the state-of-the-art Molecular Screening Shared Resources facility at UCLA, we shall employ our assays to screen libraries of ~200,000 chemical compounds to identify inhibitors that block assembly and secretion of the T6SS. We shall counter screen the compounds identified in our primary screen to identify compounds that are non-toxic and that block the replication of F. tularensis in a human macrophage cell culture model, and we shall conduct additional studies on the most potent compounds to determine how the inhibitors function. These studies will provide vital tools for studying the role of the T6SS in pathogenicity, furnish an important proof of principle that small molecule inhibitors of T6SSs can be identified and refined, and establish a foundation for a new class of antibiotics.